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CONCLUSIONS

Im Dokument Electrospun gelatin (Seite 36-50)

The following conclusions were made based on the current study.

Gelatin was electrospun from 10M aqueous acetic acid. Average fibre diameter varies from 200 nm to 700 nm depending on the concentration of the electrospinning solution. FTIR analysis, results of mechanical characterization of the material, biological degradation tests, and observations made during the experiments indicate that electrospun gelatin can be thermally cross-linked by glucose by placing the fabrics in an oven at about 170°C.

Maximum extent of cross-linking is achieved at nearly 20% glucose content.

However, easy to handle fabrics can be obtained from gelatin-glucose blends containing up to 15% glucose. At higher glucose concentrations the material becomes impractically brittle after thermal treatment. The extent of cross-link-ing depends on glucose concentration and duration of thermal treatment. Maxi-mum extent of cross-linking is reached after nearly 3 h of thermal treatment.

Mechanical properties of the material were evaluated by tensile test and modelling. The model simulates deposition of fibres during electrospinning and tensile test of fibrous meshes in order to evaluate the elastic modulus of electro-spun materials and is effective when applied to cross-linked fabrics with bonds between both polymer molecules and at fibre interceptions. Cross-linking elec-trospun gelatin by glucose increases mechanical strength of the material. The addition of aluminium potassium sulphate further increases mechanical strength of electrospun gelatin cross-linked by glucose up to about 10% alum content.

Aging of the electrospinning solution decreases mechanical strength and fibre diameter of gelatin electrospun from 10M aqueous acetic acid.

Preliminary short-term cell culture experiments indicate that electrospun gelatin cross-linked thermally by glucose is suitable for tissue engineering applications [I].

All in all, the goals of the current work were fully achieved and a promising new material, electrospun gelatin cross-linked by glucose, was developed as a result of the work.

SUMMARY IN ESTONIAN

Glükoosiga ristsidestatud elektrospinnitud želatiin

Käesolevas doktoritöö algseks eesmärgiks oli siirdamiseks sobivate želatiini-põhiste naharakkude kasvualuste väljatöötamine, kasutades võimalikult loodus-sõbralikke lähteaineid ja meetodeid. Želatiin valiti peamiseks lähteaineks, kuna see on äärmiselt biosobiv, mitte-toksiline, kergesti töödeldav ja odav. Želatiini saadakse kollageeni, ühe sidekoe peamise koostisosa, hüdrolüüsi tulemusel.

Kollageeni ja želatiini keemiline koostis on seetõttu peaaegu identne, mis teeb želatiini eriti sobivaks lähteaineks naharakkude kasvualuste valmistamisel.

Želatiini peamiseks puuduseks võrreldes sünteetiliste lähteainetega on mitte-homogeenne koostis ja sellest tulenev füüsikaliste ja keemiliste omaduste varieeruvus, kuid antud juhul on eeliseid peetud tunduvalt kaalukamaks puu-dustest.

Elusorganismis koosneb normaalne rakuväline keskkond põhimõtteliselt kokkukeerdunud nanokiudude pundardest. Sellise struktuuri teatud ulatuses jäljendamiseks valmistati kasvualused elektrospinnimise meetodil. Elektro-spinnitud želatiin on aga vees lahustuv ja mehaaniliselt nõrk. Need puudused on võimalik ületada želatiinimolekulide ristsidestamise teel. Varasemalt on selleks otstarbeks kasutatud mitmeid kalleid ja vähem või rohkem toksilisi keemilisi ristsidestajaid. Käesolev dissertatsioon põhineb avastusel, et elektrospinnitud želatiini on võimalik ristsidestada glükoosi abil. See avastus tekitas mitmeid küsimusi – kui palju glükoosi peaks želatiinile lisama soovitud tulemuse saamiseks, kui kaua peaks želatiinikangast kuumutama, kuidas ristsidestamine mõjutab materjali omadusi, kas glükoosiga ristsidestatud želatiinikangas on sobiv regeneratiivses meditsiinis kasutamiseks jne. Käesolev dissertatsioon koos selle aluseks olevate artiklitega [I–III] annab vastuse mitmetele prakti-listele küsimustele.

Suur osa dissertatsioonist on pühendatud glükoosiga ristsidestatud elektro-spinnitud želatiinikanga mehaanilistele omadustele, kuna just need mängivad võtmerolli paljudes rakendustes, sealhulgas biomeditsiinis. Samas on just žela-tiini mehaaniline nõrkus olnud tema laiema kasutamise üheks peamiseks takistuseks ja on väga oluline keskenduda nende meetodite arendamisele, mis aitaksid želatiinikiude tugevamaks muuta. Lisaks on biopolümeersete kiudude mehaanilised omadused küllaltki komplekssed ja mõjutatud muuhulgas lähte-aineks oleva želatiini kvaliteedi, valmistamisprotsessi parameetrite, lahusti omaduste, niiskuse, kiu läbimõõdu jms poolt. Kõigi nende mõjurite tõttu varieerub želatiinikiudude kirjanduses toodud elastsusmoodul äärmiselt suures ulatuses, kirjanduses leiduvaid tulemusi on omavahel väga raske võrrelda ja katsete korratavus ei ole hea, mis tekitab palju segadust. Seega on äärmiselt tähtis uurida elektrospinnitud želatiini mehaanilisi omadusi piisavalt suure detailsuse astmega, et oleks võimalik korratavalt valmistada kindlate mehaani-liste omadustega kangaid. Eeltoodust tulenevalt on käesoleva uurimuse

peamiseks eesmärgiks valmistada ja iseloomustada elektrospinnitud glükoosiga termiliselt ristsidestatud želatiinikangaid.

Töö tulemusena leiti, et želatiini ning želatiini, glükoosi ja teiste lisandite segusid on võimalik elektrospinnida 10 M äädikhappe vesilahusest. Keskmised kiudude läbimõõdud jäävad vahemikku 200–700 nm olenevalt želatiini ja lisandite kontsentratsioonist elektrospinnimise lahuses. Kangataolist, kergesti käsitletavat materjali on võimalik saada kuni 15% glükoosi sisaldavatest segu-dest. Suurema glükoosisisalduse juures muutub materjal pärast kuumutamist väga hapraks.

FTIR analüüs, želatiinikanga mehaaniliste omaduste määramise katsete tulemused, lagundamiskatsed ja katsete käigus tehtud tähelepanekud kinnitasid seda, et glükoos tõepoolest ristsidestab kiude moodustavaid želatiinimolekule, kui ta paigutada ahju umbes 170 °C juures. Seeläbi muutub želatiinikangas lahustumatuks ja mehaaniliselt palju tugevamaks. Ristsidestumise määr sõltub glükoosikontsentratsioonist ja kuumutamise kestvusest. Maksimaalne rist-sidestumise ulatus saavutatakse umbes 3 tunni kuumas ahjus hoidmise järel.

Želatiinikanga mehaanilisi omadusi iseloomustati tõmbekatse ja modelleeri-mise teel. Mudel simuleerib esiteks kiudude kogunemist elektrospinnimodelleeri-mise ajal, teiseks kiududest koosneva kanga tõmbekatset eesmärgiga määrata kiumaterjali elastsusmoodul. Mudel on eriti sobiv rakendamiseks ristsidestatud kiudmater-jalidele, mille puhul on keemilised sidemed mitte üksnes kiude moodustavate želatiinimolekulide vahel, vaid seotud on ka kiud omavahel kiudude ristumis-kohtades. Leiti, et glükoosiga ristsidestamine suurendab oluliselt elektro-spinnitud želatiini elastsusmoodulit. AlK(SO4)2 lisamine muudab glükoosiga ristsidestatud elektrospinnitud želatiini mehaaniliselt veelgi tugevamaks. Samas elektrospinnimise lahuse vanandamine vähendab valmistatava kanga mehaa-nilist tugevust, kui lahustiks on äädikhappe vesilahus.

Rakendusuuringute tulemusena leiti, et glükoosiga ristsidestatud elektro-spinnitud želatiin on sobiv regeneratiivse meditsiini rakendusteks.

ACKNOWLEDGEMENTS

I am grateful to my supervisors.

I would like to thank all the co-authors of the publications on which the dis-sertation is based on – Martin Järvekülg, Uno Mäeorg, Karol Mõisavald, Triin Kangur, Paula Reemann, Martin Pook, Annika Põder, Külli Kingo, Viljar Jaks.

I would like to express my gratitude to colleagues and friends at the Faculty of Science and Technology and Institute of Physics.

The study was financially supported by the European Union through the European Regional Development Fund via projects “Carbon Nanotube Re-inforced Electrospun Nano-fibres and Yarns“ (3.2.1101.12-0018), “SmaCell“

(3.2.1101.12-0017), Centre of Excellence “Mesosystems: Theory and Applica-tions” (3.2.0101.11-0029), Estonian Science Foundation grant IUT2-25, Esto-nian Nanotechnology Competence Centre (EU29996).

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